In general, the invention relates to a screening method for agonists of G-protein coupled receptors (GPCRs) with prolonged or short-lived activity. More specifically, the invention is related to parathyroid (PTH) hormone or PTH-related protein (PTHrP) ligand analogs that have either more prolonged or shorter-lived activity on the PTH receptor (PTHR) than does PTH(1-34). The invention also relates PTHR ligands identified using the methods of the invention and uses of such ligands in treating disease.
GPCRs are large group of membrane receptors which, in response to activation by an agonist, activate G-proteins which then, in turn, cause activation of at least one signaling cascade, such as the cyclic AMP/protein kinase A cascade. This large groups of receptors is found in organisms ranging from bacteria to man, and are involved in, for example, hormonal, neuronal, and olfactory signal transduction.
The parathyroid hormone receptor (PTHR, SEQ ID NO: 1 for human and SEQ ID NO: 2 for rat) is the endogenous receptor for both PTH and PTH related protein (PTHrP), yet each ligand has a distinct biological function. PTH regulates calcium and phosphate homeostasis and acts as a gland-secreted endocrine hormone on target cells in bone and kidney. PTH also reduces the reabsorption of inorganic phosphate (Pi) largely through its effects on sodium-dependent phosphate transporters (NaPi-IIa and NaPi-IIc) located in renal proximal tubule (PT) cells. PTHrP regulates cell proliferation and differentiation programs in developing tissues, and is secreted and acts in a paracrine fashion within tissue primordia (Kronenberg, H. M. Ann. N.Y. Acad. Sci. 1068:1-13 (2006)).
PTH (SEQ ID NO:3) and PTHrP (SEQ ID NO:4) are most homologous in their amino-terminal (residues 1-14) signaling domains (eight amino acid identities), and show moderate homology in their 14-34 binding domains (three identities). It has been generally inferred that the fully active (residues 1-34) portions of PTH and PTHrP interact with the PTHR via largely identical mechanisms (Caulfield et al., Endocrinology 127:83-87 (1990); Abou-Samra et al., Endocrinology 125:2215-2217 (1989)). This mechanism is thought to consist of two principal components: an interaction between the carboxy-terminal binding domain of the ligand and the amino-terminal extracellular (N) domain of the receptor, and an interaction between the amino-terminal signaling domain of the ligand and the juxtamembrane (J) region of the receptor, which contains the intracellular loops and seven transmembrane helices (Hoare et al., J. Biol. Chem. 276:7741-7753 (2001); Castro et al., Proc. Natl. Acad. Sci. USA 102:16084-16089 (2005); Witelsberger et al., Biochemistry 45:2027-2034 (2006); Shimizu et al., J. Biol. Chem. 280:1797-1807 (2005); Gensure et al., Biochem. Biophys. Res. Commun. 328:666-678 (2005)). However, the extent, if any, to which the precise mechanisms of binding used by the two ligands differ remains to be determined.
In humans, PTH(1-34) (SEQ ID NO:5) has potent, bone-anabolic effects, and induces marked increases in bone mineral density and bone strength. Indeed, recombinant human PTH(1-34) is now considered to be one of the most effective treatments for osteoporosis (Tashjian and Gagel, J. Bone Miner. Res 21:354-365 (2006)). Importantly, hPTH(1-34) must be administered in a pulsatile fashion (e.g., once daily subcutaneous injection) in order for its bone-forming effects to be realized. With more prolonged administrations, as with a sustained infusion pump mechanism, PTH(1-34) exerts a net catabolic effect on bone, due to a greater activation of the bone-resorptive responses mediated by the osteoclasts, relative to the bone-forming responses mediated by the osteoblasts. The duration of exposure of the PTH receptor in bone to a PTH ligand is thus a key determinant of the overall bone-formation response achieved by that ligand, and thus its effectiveness as a treatment for osteoporosis.
Clinical studies have shown that PTHrP(1-36) (SEQ ID NO:6) can also increase bone mineral density in humans, and can do so approximately to the same extent as does PTH(1-34), although higher doses are required (Horwitz et al., J. Endocrinol. Metab. 88:569-575 (2003). Importantly, even at such higher doses, PTHrP(1-36) did not stimulate the adverse, bone resorptive and hypercalcemic responses that would be expected for equivalent doses of PTH(1-34) (Horwitz et al., J. Endocrinol. Metab. 88:569-575 (2003); Horwitz et al., J. Bone Miner. Res. 20:1792-1803 (2005); Horwitz et al., Osteoporosis Int. 17:225-230 (2006)). The difference in biological activity of the two peptides is not due merely to a difference in pharamacokinetics. A direct comparison of the two peptides using steady-state infusions methods showed that PTHrP(1-36) is markedly less efficacious than PTH(1-34) for stimulating the renal synthesis of 1,25-(OH)2 vitamin D3 (Horwitz et al., J. Bone. Mineral. Research. 20:1792-1803 (2005)).
In addition to osteoporosis, hPTH(1-34) (SEQ ID NO:5) has been shown to be effective in treating conditions of PTH deficiency, namely hypoparathyroidism. Thus, PTH(1-34) was shown to be a safe and effective alternative to calcitriol therapy and was able to maintain normal serum calcium levels without hypercalciuria in patients with hypoparathyroidism (Winer et al., J. Clin. Endocrinol. Metab. 88:4214-4220 (2003)). The peptide had to be injected at least twice daily, and the authors recognized the need in this disease for a long-acting PTH(1-34) analog (Winer et al., J. Clin. Endocrinol. Metab. 88:4214-4220 (2003).
Therefore, there exists a need in the art for PTH or PTHrP analogs that have longer- or shorter-lived actions on the PTH receptor than does PTH(1-34). There also exists a need for assays that allow one to distinguish between PTH peptides that have short-versus long-acting effects.